Manipulation of Actin Cytoskeleton by Intracellular-Targeted ROS Generation.

A method to generate small amount of reactive oxygen species (ROSs) at intracellular targeted region has great potential to manipulate the function of particular proteins. The present protocol introduces a fusion protein that consisted of firefly luciferase (FLuc), photosensitizer protein KillerRed and F-actin-targeting peptide Lifeact (Lifeact-KillerFirefly) to generate ROSs in the vicinity of F-actin and found that morphological change in F-actin structure was induced by the fusion protein after luciferin treatment. This manipulating and imaging method is of use to analyze the role of the locally generated ROSs on the function of intracellular proteins.